Method of enhancing learning rate and retention level in warm blooded animals



United States Patent W METHOD OF ENHANCDIG LEARNING RATE AND RETENTIONLEVEL 1N WARM BLGODED ANI- MALS Alvin J. Glasky, Lincolnwood, andNicholas P. Plotniholf, Lake Bluff, 11]., assignors to AbbottLaboratories, North Chicago, 111., a corporation of Illinois No Drawing.Filed May 26, 1965, Ser. No. 459,103

3 Claims. (Cl. 167-65) This invention relates to a method of stimulatingribonucleic acid synthesis and in particular to a method of improvingimpaired learning ability or impaired retention in mammals.

While it is not intended to rely upon any particular theory to explainthe therapeutic effectiveness of the method of the present invention,the hypothesis has been advanced that ribonucleic acid (RNA) is thesubstrate for memory (Hyden, H., 1955, Nucleic Acid and Proteins,Neurochemistry, pp. 222-224) since, by the rearrangement of the fourbases of ribonucleic acid, many combinations of the molecule arepossible, thereby allowing or more bits of information to be encoded.Cameron, D. E. (1963), The Process of Remembering, Brit. J. Psych. 109:325-340 reports that the administration of ribonucleic acid (RNA), bothin oral and intravenous form, at least with the former resulted in someevidence of amelioration of memory deficits in aged individuals beingstudied. The intravenous solutions produced severe shock-like reactions.

It has been found that the administration to warmblooded animals ofp-amino-N-(2-diethylaminoethyl) benzamide or its neutral or acidaddition salts results in an increased rate of learning by the animaltogether with a prolonged period of rentention of the learned behavior.It is believed that the enhanced learning and prolonged memory effect ofthese compounds may be due to their stimulation of brain ribonucleicacid synthesis. Thus, the noted compounds are useful in conditions suchas impaired learning ability or impaired retention and for thoseconditions where enhanced synthesis of cellular ribonucleic acid wouldbe beneficial.

The following examples are presented in order to more fully disclose theinvention. It should be understood, however, that the examples are notintended to limit the invention in any way.

Example I T 0 determine whether or not the noted compounds cause an invivo stimulation of a ribonucleic synthesizing enzyme, p amino N(Z-diethylaminoethyl) benzamide sulfate was administered to female rats.One day after administration mg./kg., daily by gavage) was begun, theanimals were mated. Twenty-one days later, the pregnant animals weresacrificed and brain tissue was removed from the fetuses. A nuclearaggregate enzyme was prepared from the brain tissue and found toincorporate a radioactively labeled RNA precursor (nucleosidetriphosphate) into RNA. Incorporation of nucleoside triphosphate intoRNA was not observed with a nuclear aggregate enzyme derived fromcontrol animals (saline treated) not pretreated withp-amino-N-(Z-diethylamino- 3,321,369 Patented May 23, 1967 ethyl)benzamide sulfate. The procedure was repeated with the acid sulfate andwith p-amino-N-(Z-diethylaminoethyl) benzamide hydrochloride. The sameresults were obtained in each case.

Thus, it has been shown that the noted compounds cause in vivostimulation of an RNA synthesizing enzyme.

Example II To assay the increased synthesis of RNA in the intact ratbrain, a radioactively labeled precursor of nucleic acid (orotic acid-Cwas injected intracranially at various times after the intraperitonealadministration of p-amino- N-(Z-diethy-laminoethyl) benzamide sulfate,acid sulfate or hydrochloride. The animals were later sacrificed and theamount of RNA synthesized in vivo was measured, i.e., the amount ofradioactive precursor converted into nucleic acid was measured. A markedstimulation in the amount of RNA synthesized in vivo as early as 15-30minutes after administration of the drug was found. The productsynthesized was identified as RNA.

As can be seen, p-amino-N-(Z-diethylaminoethyl) benzamide sulfate, acidsulfate or hydrochloride caused an in vivo stimulation of RNA synthesis.

Example III A nuclear aggregate enzyme was isolated from brain tissuederived from non-treated adult rats. The enzyme was inactive when testedin the normal assay procedure. However, whenp-amino-N-(2-diethylaminoethyl) benzamide sulfate, acid sulfate orhydrochloride was added to the system, in vitro, the enzyme activity wasmarkedly stimulated. Thus, an in vitro stimulation of an RNAsynthesizing system was accomplished.

Synthesis of RNA is essential to the functioning of all cells and iscatalyzed by the enzyme, RNA polymerase. Essentially, in vitro and invivo data have been obtained which show the stimulation of fetal ratbrain RNA synthesis by the administration ofp-amino-N-(Z-diethylaminoethyl) benzamide sulfate, acid sulfate orhydrochloride through an apparent activation of brain RNA polymerase.

The behavioral effects resulting from administration of the noted drugswere evaluated on a modification of the Cook-Weidley apparatus (L. Cookand E. Weidley, Ann. N.Y. Acad. Sci., 66, 740, 1957). Essentially, suchapparatus consists of a chamber with a grid flooring and an escapeplatform outside the chamber. Several different training schedules forconditioned avoidance were employed. Each training trial consisted of 30seconds of buzzer and electric shock stimulation to the rat in thechamber. Subsequent test trials (acquisition trials) consisted of 15seconds in the chamber without any stimulation followed by 10 seconds ofbuzzer stimulation and culminated by 5 seconds of buzzer plus shockstimulation. The time from entrance into the apparatus until the ratjumps out is recorded as the escape time. Five or ten test trials orretention trials were employed each day. Retention trials were conductedby testing the animals escape response in the test chamber during a30-second time period without stimulation of any kind. The test sequencefor any given trial was terminated upon successful completion of thetask.

Retention Trials y sec.

amide/ .lkg. I.P.

Retention Controls Test Trials y sec.

8350838522 md ifi d ad id d Retention Trials y sec.

2807585725 om44-L7 8 &6 7 5 1111 Retention Trials (D ay 2), sec.

d-N-a-dimethylphenethyl amine hydrochloride, 1.0 mgJkg. I.P.

Test Trials y sec.

3285875722 QYQLRMQWQQO 2 11 111 1 Example VI 11. Oil. 7

ONE T RAININ G T RIAL Controls Retention Trials sec.

Test Trials Example IV o-Chloro-p-amino N- (diethylaminoethyDbenzamide,20 mg Test Trials y sec.

ONE TRAINING TRIAL Retention Trials y sec.

p-Aminobenzoyl diethylamino ethanol, 20 mg./ kg. LP.

Test Trials y sec.

Example V p-Amino-N-(2 diethylaminoethybbenzamide HCl, 20 mg./kg. LP.

Test Trials y sec.

ONE TRAINING TRIAL n l a u T n O 1 0 3 0 90588969 1 m 4 m m e s m R V. mmw 7255802002 6 a 9 0L&7 & 6 .U 111111HM 1 mm T 0 t 1 T 1 0688166668 2 1H 8 m .ms mu mm m 1 a an) i n1. .3 1 t t Mm GT n n R D .1 mk 8 1%7508325737 4 Wm m 4422 25 &220 L t 1 1111111111 I m 3 1 .1 an... m a 2 TTrial 1 Drug administered intraperitoneally minutes prior to trainingtrial. 2 Average escape time for groups of 6 rats.

Trial 5th day 4th day 3rd day 2nd day Test Trials 1st day Dose, mgJkg.

rats

mino-N-(2-diethylaminoethyl) benzamide-HCl] injected end of test periodeach day.

Example VIII FIVE TRAINING TRIALS Number of 1 Five training trials.

One injection [p-amino-N- 2nd Day, see. 3rd Day, sec. 4th Day, sec. 5thDay, sec. 2nd Week, See. 3rd Week, see. 8th Week, sec.

, 4.2iOA; Controls, 9.9i1.0.

Group Group A: Drug [p-a Group B:

of 6 rats.

Dose [p-aminoN-(2- diethylamino ethyl) benza.mide-HC1]I.P.

20 mgJlrrr mg. q Controls ./kg., 4.45:0.4; 50 mgJkg.

t Prug administered intraperitoneally 15 minutes prior to training 2Average escape time for groups Number of rats 1 Drug administeredintraperitoneally 15 minutes prior to first training trial. 1 Averageescape time for group of 12 rats in 5 test trials. Overall averages: 20mg Example IX p-Amino-N-(Z-diethylp-Arn ino-N-(2-diethyl aminoethyl)benz- Controls annnoethyl) benzamide, 20 mgJkg. I .P. amide-H 804, 20mgJkg. Tn'al LP.

Test Trial Retention Test Trial Retention Test Trial Retention (Day 1)Trial (Day (Day 1), Trial (Day (Day 1), Trial (Day sec. 2), sec. sec.2), sec. sec. 2), sec.

1 All test animals learned to escape within fifteen seconds.

Example X MIDE-HCI, ONE HOUR PRETREATMEN 20 mgJkg. 50 mgJkg. ControlsTest Trial Retention Test Trial Retention Test Trial Retention Trial(Day 1) Trial (Day (Day 1), Trial (Day (Day 1), Trial (Day sec. 2), sec.sec. 2), sec. sec. 2), sec.

1 All test animals learned to escape within fifteen seconds.

The foregoing examples show a markedly shortened escape time in both thetest and retention trials in rats treated withp-amino-N-(Z-diethylaminocthyl) benzamide or its neutral or acidaddition salts over a dosage range of 10 to 50 mg./kg. intraperitoneallyand to 100 mg./ kg. orally. Pretreatment of the rats during the trainingtrials both orally and intraperitoneally resulted in markedly increasedacquisition rates in comparison to control rats treated with salinesolution. Post-treatment of rats following training resulted in markedlyprolonged retention of learned performance, up to eight weeks followingdrug administration. Comparable studies carried out withp-aminobenzoyldiethylaminoethanol, o-chloro-p-amino-N(diethylaminoethyl) benzamide, d-N-a-dimcthylphenethylamine, and2-dimethylaminoethanol did not show any significant increase inacquisition rate or retention level of avoidance response compared tosaline-treated animals.

All forms of p-amino-N-(Z-diethylaminoethyl) benzamide; that is, base,sulfate, acid sulfate, and hydrochloride salts, were found to beefiective in accelerating acquisition rates of avoidance responses aswell as increasing retention levels of these responses over prolongedperiods of time. The optimal dose in all forms was found to be 20mg./kg. The minimum effective dose was found to be 10 mg./kg.

On the basis of the biochemical and behavioral effects resulting fromthe administration of the noted compounds, therapeutic utility has beendemonstrated in the enhancement of learning performance and retention;acceleration of normal cellular processes and functions dependent onribonucleic acid metabolism; all clinical conditions Wherein enhancedribonucleic synthesis would be beneficial, including but not limited to,deceleration of degenerative aging processes, accelerated tissue repairand accelerated synthesis of cellular components; and psychiatricclinical conditions where performance is impaired.

Lactose In the foregoing examples, the compounds were administered bydispersing the compounds in a liquid carrier. It should be understood,however, that the present invention contemplates providing the activecompound in the form of a granulation, tablets, capsules, elixirs,amulsions, and other dosage forms well known to the art.

Example XI In illustration of the tablet dosage form, the following is aformula for 100 tablets:

The tablets are prepared by mixing the acid-addition salt top-amino-N-(Z-diethylaminoethyl) benzamide with lactose, moistening andscreening. The mixture is granulated with a corn starch paste made bydissolving 3 grams of corn starch in cc. of water. The granulatedmixture is then dried throughly. To the granulation is added the talc,magnesium stearate, and the remainder of the corn starch. After thoroughmixing, the tablets are compressed in a conventional tabletting machine.Each tablet will provide 200 mg. of active ingredient.

Example XII A suitable capsule mixture formula for capsules is asfollows:

Grams Hydrochloric acid, acid-acid addition salt of p-amino-N-(Z-diethylaminoethyl) benzamide 10 The ingredients are thoroughlymixed and placed in a gelatin capsule. Each capsule will provide 100milligrams of active ingredient.

Example XIII An aqueous solution for injection can be made by dissolving1 gram of p-amino-N-(Z-diethylaminoethyl) benzamide in 500 ml. ofsterile water. The solution is then filled into 5 ml. ampoules which aresealed and sterilized. Each ampoule will contain mg. of activeingredient.

The compounds of this invention have been found to be etfective whenadministered orally to rats in the dosage range of from to 100 mg./ kg.but can be administered orally to other warm-blooded animals in thedosage range of about 100 to 300 mg. per day. A convenient method is toadminister the total daily dose in three equal dosage forms. In smalleranimals other than rats, usually about half the above dose will sufiice,i.e., from about mg. to about mg. per day.

Others may practice the invention in any of the numerous ways which willbe suggested by this disclosure to one skilled in the art. All suchparticles of the invention are considered to be a part thereof providedthey fall within the scope of the appended claims.

What is claimed is: I

1. The method of enhancing learning rate and retention level impairmentin warm-blooded animals which comprises administering to a warm-bloodedanimal having said impairment from about 10 to 300 milligrams daily ofan active ingredient selected from the group consisting ofp-amino-N-(Z-diethylaminoethyl) benzamide and its neutral or acidaddition salt.

2. The method as defined in claim 1 wherein the active ingredientcomprises the sulfuric acid, acid addition salt ofp-amino-N-(Z-diethylarninoethyl) benzamide.

3. The method as defined in claim 1 wherein the active ingredientcomprises the hydrochloric acid, acid addition salt ofp-amino-N-(Z-diethylaminoethyl) benzamide.

References Cited by the Examiner American Drug Index, (1962), page 626,J. B. Lippencott Company, Philadelphia and Montreal.

Merck Index, (7th edition, 1960) page 853.

ALBERT T. MEYERS, Primary Examiner.

JULIAN S. LEVIT'I, Examiner.

N. G. MANN, STANLEY J. FRIEDMAN,

Assistant Examiners.

1. THE METHOD OF ENHANCING LEARNING RATE AND RETENTION LEVEL IMPAIRMENTIN WARM-BLOODED ANIMALS WHICH COMPRISES ADMINISTERING TO A WARM-BLOODEDANIMAL HAVING SAID IMPAIRMENT FROM ABOUT 10 TO 300 MILLIGRAMS DAILY OFAN ACTIVE INGREDIENT SELECTED FROM THE GROUP CONSISTING OFP-AMINO-N-(2-DIETHYLAMINOETHYL) BENZAMIDE AND ITS NEUTRAL OR ACIDADDITION SALT.